Sequence: bcl-2-like protein 11 isoform 18 [Homo sapiens] Description ..    BCL2L11 BCL2-like 11 (apoptosis facilitator) [ Homo sapiens ], BIM Protein Family: Bim protein N-terminus Gene:BCL2L11

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University of New Mexico Assay Overview:
Assay Support: NIH 1X01 MH079850-01
HTS to identify small molecule regulators of Bcl-2 family protein interactions
PI: Larry Sklar, Ph.D.
Assay Implementatiion: Peter Simons Ph.D, Susan Young MS, Anna Waller Ph.D, Mark Carter MS

Assay Background and Significance:

This report summarizes the series of assays used to identify small molecule regulators of Bcl-2 family protein interactions. One arm of apoptosis is regulated by the balance of anti-apoptotic and pro-apoptotic Bcl-2 family members. The binding of fluorochrome-conjugated BH3 peptides (in this case, Bim (also known as BCL2-like 11)) to Bcl-2 family members provides the basis for construction of a fluorescence-based assay amenable to flow cytometry high throughput screening for small molecule regulators of these interactions. This is a multiplexed assay to identify small molecule regulators of protein interactions between the BH3 peptide of Bim and the following six Bcl-2 family members: Bcl-XL, Bcl-W, Bcl-B, Bfl-1, and Mcl-1 and Bcl-2 (the eponymous founding member of the Bcl-2 family).

The present report assembles the list of compounds that showed activity across all Bcl protein assays.

This project lead to a probe for Bcl-B:
(SID / CID / ML#)
124756688 / 53301938 / ML258

Each component of the multiplex assay consists of a glutathione labeled bead, a GST Bcl-fusion protein target (six total, supplied by project collaborator), and a fluorescent peptide probe, F-Bim (FITC-Axh-DMRPEIWIAQELRRIGDEFNAYYAR-OH; Commonwealth Biotech, USA). Bead sets are coated with individual GST-conjugated Bcl-2 proteins in HPSMTB buffer (30milliM HEPES, 100milliM KCl, 20milliM NaCl, 1milliM MgCl(2), 0.01% Tween-20, 0.1% BSA) and incubated overnight at 4 degrees C.

The mulitplex is constructed by using beads for each protein target that have been labeled with varying intensities of red color, so that each assay is built on a unique bead set, and each bead set is associated with a unique optical address. Beads are first washed in HPSMTB buffer for 20 minutes before adding the appropriate GST-Bcl fusion protein. The bead sets (ThermoFisher Scientific product numbers XPR-1687-XPR-1696), have similar size (~ 4 micron diameter) and are distinguished by distinct emission characteristics at 665 +/-10 nm with excitation at 635 nm. Thus, GST-Bfl-1 might be noncovalently coated onto red level 1 beads, GST-Bcl-XL onto red level 2 beads, etc. The 6 bead sets (each with bound protein) and uncoated beads (see below) are first centrifuged separately, then combined and centrifuged again, and finally diluted just before loading into 384-well plates to minimize bead-protein dissociation before the assay begins.

The assay is conducted in 384-well microplates in a total assay volume per well of 10.1 microliters (5 microL of bead mixture, 0.1 microL of test compound, and 5 microL of 100nM F-Bim in HPSMTB). Test compound concentration is 10 microM. Controls, which contain bead mixture and F-Bim but no test compound, are located in columns 1 and 2 on each plate. Plates are placed horizontal axis on rotators and incubated for 1-2 hours at 4 degrees C.

A glutathione-only bead set control (no associated GST-protein) is incorporated into each well as a fluorescence scavenger to determine inherent fluorescent properties (at 530 nm emission) of the test compounds. Specificity of F-Bim binding is determined with a Positive Control using a block of the F-Bim Fluor with a non-fluoresceinated F-Bim peptide. The F-Bim blocking control is run daily as a separate single tube assay using F-Bim at 5 microM.

Sample analysis is conducted with the HyperCyt(R) high throughput flow cytometry platform. The HyperCyt system interfaces a flow cytometer and autosampler for high-throughput microliter-volume sampling from 384-well microtiter plates. Flow cytometric data of light scatter and fluorescence emission at 530 +/- 20 nm (FL1) and 665 +/- 10 nm (FL8) are collected on a Cyan Flow Cytometer (Dako). Analysis is performed using time-resolved acquisition into a single data file and analysis using IDLeQuery software to merge the flow cytometry data files with compound worklist files generated by HyperSip software. The raw data are parsed in IDLeQuery to produce annotated fluorescence summary data for each well. The parsed data are then processed through an Excel template file constructed specifically for the assay to segregate data for each target and the fluorescence scavenger in the multiplex. Gating based on forward scatter (FS) and side scatter (SS) parameters is used to identify singlet bead populations. Gating based on FL8 emission distinguishes the beads coated with different proteins, and the median fluorescence per bead population is calculated.

Summary and Sequence of Assays Performed

HTS Bim-Bcl-2 AID # 950
Method: multiplex flow cytometry bead-based fluorescent ligand binding competition assay
Test concentration: 10 microM
Activity criterion: >40% change in % inhibition
# compounds evaluated: 194,830
# active compounds: 116

HTS Bim-Bcl-B AID # 951
Method: multiplex flow cytometry bead-based fluorescent ligand binding competition assay
Test concentration: 10 microM
Activity criterion: >40% change in % inhibition
# compounds evaluated: 194,830
# active compounds: 142

HTS Bim-Bcl-w AID # 952
Method: multiplex flow cytometry bead-based fluorescent ligand binding competition assay
Test concentration: 10 microM
Activity criterion: >40% change in % inhibition
# compounds evaluated: 194,830
# active compounds: 47

HTS Bim-Bcl-xL AID # 1007
Method: multiplex flow cytometry bead-based fluorescent ligand binding competition assay
Test concentration: 10 microM
Activity criterion: >40% change in % inhibition
# compounds evaluated: 194,830
# active compounds: 82

HTS Bim-Bfl-1 AID # 1008
Method: multiplex flow cytometry bead-based fluorescent ligand binding competition assay
Test concentration: 10 microM
Activity criterion: >40% change in % inhibition
# compounds evaluated: 194,830
# active compounds: 237

HTS Bim-Mcl-1 AID # 1009
Method: multiplex flow cytometry bead-based fluorescent ligand binding competition assay
Test concentration: 10 microM
Activity criterion: >40% change in % inhibition
# compounds evaluated: 194,830
# active compounds: 196

Dose Response Bim_Bcl-w AID # 1330
Method: multiplex flow cytometry bead-based fluorescent ligand-binding competition assay
Test concentration: eight point concentration curve from 10 nanoM to 100 microM
Activity criterion: EC50 less than 10 microM and magnitude of response greater than 40%
# compounds evaluated: 834
# active compounds: 18

Dose Response Bim_Bcl-b AID # 1327
Method: multiplex flow cytometry bead-based fluorescent ligand-binding competition assay
Test concentration: eight point concentration curve from 10 nanoM to 100 microM
Activity criterion: EC50 less than 10 microM and magnitude of response greater than 40%
# compounds evaluated: 834
# active compounds: 6

Dose Response Bim_Bfl-1 AID # 1320
Method: multiplex flow cytometry bead-based fluorescent ligand-binding competition assay
Test concentration: eight point concentration curve from 10 nanoM to 100 microM
Activity criterion: EC50 less than 10 microM and magnitude of response greater than 40%
# compounds evaluated: 834
# active compounds: 97

Dose Response Bim_Bcl-x AID # 1322
Method: multiplex flow cytometry bead-based fluorescent ligand-binding competition assay
Test concentration: eight point concentration curve from 10 nanoM to 100 microM
Activity criterion: EC50 less than 10 microM and magnitude of response greater than 40%
# compounds evaluated: 834
# active compounds: 6

Dose Response Bim_Bcl-2 AID # 1328
Method: multiplex flow cytometry bead-based fluorescent ligand-binding competition assay
Test concentration: eight point concentration curve from 10 nanoM to 100 microM
Activity criterion: EC50 less than 10 microM and magnitude of response greater than 40%
# compounds evaluated: 834
# active compounds: 9

Dose Response Bim_Mcl-1 AID # 1329
Method: multiplex flow cytometry bead-based fluorescent ligand-binding competition assay
Test concentration: eight point concentration curve from 10 nanoM to 100 microM
Activity criterion: EC50 less than 10 microM and magnitude of response greater than 40%
# compounds evaluated: 834
# active compounds: 3

Profiling Assay for GST-GSH interaction in multiplex bead-based assays AID # 1324
Method: multiplex flow cytometry bead-based fluorescent ligand-binding competition assay
Test concentration: eight point concentration curve from 10 nanoM to 100 microM
Activity criterion: EC50 less than 10 microM and magnitude of response greater than 20%
# compounds evaluated: 834
# active compounds: 19

Dose Response Bim_Bcl-w compound powder confirmation AID # 2081
Method: multiplex flow cytometry bead-based fluorescent ligand-binding competition assay
Test concentration: eight point concentration curve from 10 nanoM to 100 microM
Activity criterion: EC50 less than 10 microM and magnitude of response greater than 40%
# compounds evaluated: 42
# active compounds: 5

Dose Response Bim_Bcl-B compound powder confirmation AID # 2077
Method: multiplex flow cytometry bead-based fluorescent ligand-binding competition assay
Test concentration: eight point concentration curve from 10 nanoM to 100 microM
Activity criterion: EC50 less than 10 microM and magnitude of response greater than 40%
# compounds evaluated: 42
# active compounds: 9

Dose Response Bim_Bfl-1 compound powder confirmation AID # 2080
Method: multiplex flow cytometry bead-based fluorescent ligand-binding competition assay
Test concentration: eight point concentration curve from 10 nanoM to 100 microM
Activity criterion: EC50 less than 10 microM and magnitude of response greater than 40%
# compounds evaluated: 42
# active compounds: 23

Dose Response compound powder confirmation Bim_Bcl-xl AID # 2084
Method: multiplex flow cytometry bead-based fluorescent ligand-binding competition assay
Test concentration: eight point concentration curve from 10 nanoM to 100 microM
Activity criterion: EC50 less than 10 microM and magnitude of response greater than 40%
# compounds evaluated: 42
# active compounds: 0

Dose Response Bim_Bcl-2 compound powder confirmation AID # 2075
Method: multiplex flow cytometry bead-based fluorescent ligand-binding competition assay
Test concentration: eight point concentration curve from 10 nanoM to 100 microM
Activity criterion: EC50 less than 10 microM and magnitude of response greater than 40%
# compounds evaluated: 42
# active compounds: 6

Dose Response Bim_Mcl-1 compound powder confirmation AID # 2086
Method: multiplex flow cytometry bead-based fluorescent ligand-binding competition assay
Test concentration: eight point concentration curve from 10 nanoM to 100 microM
Activity criterion: EC50 less than 10 microM and magnitude of response greater than 40%
# compounds evaluated: 42
# active compounds: 0

Abbreviations: nm for nanometer, nanoM for nanomolar, microM for micromolar, milliM for millimolar, microL for microliter

The Gene ID utilized to describe the target covers the actual fluorescently tagged Bim protein used as the ligand for this multiplex project: F-Bim (FITC-Axh-DMRPEIWIAQELRRIGDEFNAYYAR-OH). There is no protein GI for this short peptide sequence.

Grant Number: 1X01 MH079850-01